During FY17 we accomplished the following: 1) Completed one phase of analysis of IGCR1-deleted IgH alleles. A manuscript describing this work was submitted for publication. 2) We continued analyses of cell lines generated during FY16 that altered additional CTCF-binding sites (beyond IGCR1) in the proximal VH region. We assayed changes in histone modifications caused by deletion of single or multiple CTCF sites using ChIP. We also used deep sequencing to identify changes in VH gene utilization during VDJ recombination in pro-B cells deleted of specific CTCF-binding sites in the proximal VH region. 3) We generated cell lines in which individual CTCF sites within IGCR1 were inverted. In these cells CTCF binding should be unaffected, permitting us to determine the role of CTCF orientation on regulating chromatin looping and VDJ recombination. 4) We found that DST4.2, a DH gene segment that is located 70kb 5 of DFL16.1 and rarely used in DH recombination on WT IgH alleles, recombined at robust levels on IGCR1-mutated IgH alleles. All observed recombination events occurred by deleting intervening DNA. This is most consistent with the recently proposed model of RAG1/2 tracking from the JH-associated recombination center. However, DST4.2 also contains a 5 RSS that could be used for inversional recombination, but is not utilized. To test the possibility that this RSS is functionally impaired we are generating several plasmids in which 5 or 3 RSSs associated with DH gene segments will be tested independently with a JH1 RSS for recombination efficiency.